molecules

Article

A New UPLC-MS/MS Method for theCharacterization and Discrimination ofPolysaccharides from Genus Ephedra Based onEnzymatic Digestions

Yong-Gang Xia, Tian-Long Wang, Li-Ming Sun, Jun Liang, Bing-You Yang and Hai-Xue Kuang *

Key Laboratory of Chinese Materia Medica, Heilongjiang University of Chinese Medicine,Ministry of Education, Harbin 150040, China; yonggangxia@163.com (Y.-G.X.); wtl890322@163.com (T.-L.W.);llimingsun@163.com (L.-M.S.); lliangjunn@163.com (J.L.); ybywater@163.com (B.-Y.Y.)* Correspondence: hxkuang56@163.com; Tel.: +86-451-8276-7188

Received: 29 October 2017; Accepted: 15 November 2017; Published: 17 November 2017

Abstract: Ephedra sinica polysaccharides have been reported to possess important activities,so quality evaluation of polysaccharides from the genus Ephedra is urgent. In this study, enzymaticdigestions were performed to establish multiple saccharide fingerprints by ultra-performance liquidchromatography with electrospray ionization triple quadrupole linear ion trap mass spectrometry(UPLC-ESI-TQ-MS/MS) based on a multiple-reaction monitoring in negative mode. Under optimumUPLC-ESI--TQ-MS/MS conditions, excellent separation and quantification of 21 constituents wereachieved within 20 min on a solid core column with a 1.6 µm particle using pre-column derivatizationwith a PMP reagent. This method, coupled with enzymatic digestions and principal componentanalysis, has been successfully applied to characterize and discriminate Ephedra polysaccharidesattributed to different species and plant parts. The results suggest that the proposed analytical strategycould achieve a quality evaluation of plant polysaccharides from traditional Chinese medicines.

Keywords: UPLC-MS/MS; polysaccharides; genus Ephedra; enzymatic digestion

1. Introduction

Mahuang is a famous traditional Chinese medicine (TCM) that has been used for thousandsof years for the treatment of allergies, asthma, pneumonia, bronchitis, hay fever, and colds [1].According to the 2015 edition of the Chinese pharmacopoeia, Mahuang is dry stems of Ephedra sinicaStapf, E. intermedia Schrenket C. A. Mey. and E. equisetina Bge., while Radix Ephedrae is recorded to beanother different TCM, which is the dry roots or rhizoma of E. sinica and E. intermedia in the officialpharmacopoeia of China [2]. Recently, some reports have demonstrated that the stems of E. sinicacontain a high amount of polysaccharides, ranging from 3% to 5% of the total weight [3,4]. In addition,E. sinica stem polysaccharides showed obviously immunosuppressive effects by a carbon clearancetest, delayed type hypersensitivity reaction, and humoral immune response in vivo [4–6]. However,so far there is no published information on discrimination of Ephedra polysaccharides from differentspecies and plant parts. Therefore, it is essential to develop an effective method for quality evaluationof Ephedra polysaccharides.

A variety of chromatographic techniques such as gas chromatography (GC) [7], capillaryelectrophoresis (CE) [8], high-performance liquid chromatography (HPLC) [9,10], and high-performanceanion exchange chromatography (HPAEC) [11,12] were previously considered as useful tools for thedetermination of carbohydrates. Although existing techniques are increasingly mature and graduallyimproving, they still face many problems and challenges. The limit of quantification of the GC,HPLC-UV, and CE-UV method is too restrictive and cannot be used in trace quantification of

Molecules 2017, 22, 1992; doi:10.3390/molecules22111992 www.mdpi.com/journal/molecules

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carbohydrates. HPAEC can achieve more satisfactory separation than HPLC techniques, but theepimerization and degradation can be issues at high pH [11,12]. Except for HPAEC, most of the LCand CE techniques often use labeling with either fluorescence or UV tags for enhanced detection.The reagent 1-phenyl-3-methyl-5-pyrzolone (PMP) has been widely used for composition analysis ofplant polysaccharides [3].

Carbohydrates encompass a number of homologues with similar structures, so the analysisof carbohydrates inevitably requires high-resolution separation techniques. With the progress ofchromatographic techniques, ultra-performance liquid chromatography (UPLC) was developed witha sub-2 µm diameter, which shortened the analysis time and provided high resolution and sensitivityseparation, especially tandem mass spectrometry (MS/MS). Although UPLC-MS/MS have beenapplied to analyze plant polysaccharides, most reports are limited to several monosaccharides [13,14].

In this study, UPLC with triple quadrupole linear ion trap mass spectrometry (TQ-MS/MS)was firstly developed for simultaneous analysis of 21 carbohydrates and one degradation product(5-HMF, 5-hydroxymethyl furfural) based on a pre-column PMP derivatization. Elution of all eightneutral sugars, two uronic acids, three amino sugars, and two acetyl amino sugars, was thenused to evaluate the performance of this method. Finally, UPLC-ESI−-TQ-MS/MS coupled withenzymatic digestions and principal component analysis (PCA) has been successfully applied toanalyze and characterize 20 Ephedra polysaccharide samples attributed to different species and plantparts. The results showed that the proposed analytical methods provided reasonable insight andguidance for quality evaluation of plant polysaccharides from the genus Ephedra.

2. Experimental

2.1. Materials and Reagents

E. sinica, E. intermedia, and E. equisetina were purchased in September 2012 from Datong,Shanxi Province, China and identified by Professor Zhen-Yue Wang of Heilongjiang University ofChinese Medicine. The voucher specimens (20120905) were deposited at the herbarium of HeilongjiangUniversity of Chinese Medicine, Harbin, China.

D-galacturonic acid (GalUA), D-glucuronic acid (GlcUA), D-fucose (Fuc), L-rhamnose (Rha),D-glucose (Glc), D-galactose (Gal), D-mannose (Man), L-arabinose (Ara),D-ribose (Rib), N-Acetyl-D-(+)-glucosamine (GlcNAc), N-Acetyl-D-(+)-galactosam-ine (GalNAc), D-glucosamine hydrochloride (GlcN),D-galactosamine hydrochloride (GalN), D-mannosamine hydrochloride (ManN), 5-hydroxymethylfurfural (5-HMF), lactose, maltotriose, maltotetraose, maltopentaose, maltohexaose, and maltoheptaosewere purchased from Sigma- Aldrich (St. Louis, MO, USA). D2-Glc, 1-phenyl-3-methyl-5-pyrazolone(PMP), ammonium formate (AF), and formic acid (FA) were also obtained from Sigma-Aldrich. Pectinase,α-amylase, and cellulose were purchased from Sigma-Aldrich; xylanase and endo-1,3-β-glucanase wereobtained from Megazyme. Water was obtained from a Milli-Q purification system (Millipore, Bedford,MA, USA). All other chemicals were of the highest analytical grade.

2.2. Preparation of Polysaccharides from Genus Ephedra

The dried sample materials (100 g) were extracted three times with 10-fold volume of distilledwater under reflux for 3 h each time. The filtrate of the obtained extract was condensed under a vacuumto syrup and precipitated with 95% ethanol added to a final concentration of 80%. After standing for 24 hat 4 ◦C, the precipitate was collected and washed with anhydrous ethanol, then dried. The residue wasre-dissolved in water, and then, after centrifugation (4000 rpm for 15 min), the supernatant was dialyzed(cut-off Mw 3500 Da) against tap water for 48 h and distilled water for 48 h and lyophilized to yieldcrude polysaccharides. The crude polysaccharides, which were prepared in duplicate, were obtainedfor further analysis.

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2.3. Specific Enzymatic Digestion of Ephedra Polysaccharides

Polysaccharide solutions (5 mg/mL, 200 µL) were mixed with specific enzymes (the finalconcentrations of pectinase, endo-1,4-β-xylanase, cellulose, β-(1→3)-D-glucanase and α-amylase were30 U/mL, 10 U/mL, 0.3 U/mL, 0.2 U/mL and 30 U/mL, respectively) in a 1.5-mL centrifuge tubeand digested overnight (≥15 h) at 50 ◦C. Then the mixtures were heated at 80 ◦C for 20 min to stopthe enzymatic digestion. After centrifugation (12,000 rpm/min) at room temperature for 20 min,the supernatants were evaporated to dryness with a nitrogen evaporator at 70 ◦C and then used forderivatization with PMP. Moreover, the blank control was treated as described above without enzymes.

2.4. Derivatization with PMP Reagent

PMP derivatization of carbohydrates was carried out as described previously with propermodifications [3]. Briefly, PMP was prepared in methanol at a final concentration of 0.5 M. One hundredmicroliters of each individual standard monosaccharide and oligosaccharide, mix standard solutions,and the hydrolyzed polysaccharide samples were placed in 1.5-mL centrifuge tubes. Then, 0.5 M PMP(100 µL) and ammonia (200 µL) were added. D2-Glc as an internal standard (2 M) was added to eachsample (2 µL). Each mixture was allowed to react for 20 min in a 70 ◦C water bath, then cooled to roomtemperature and neutralized with 200 µL of formic acid. The solution was directly filtered througha 0.22-µm membrane and diluted with deionized water before UPLC-TQ-MS/MS analysis.

2.5. UPLC Apparatus and Conditions

An ACQUITY ultra-performance liquid chromatographic system (Waters, Milford, MA, USA)was applied. The chromatographic separation was conducted on a cortecs UPLC C18 column(1.6 µm, 2.1 × 100 mm) equipped with a cortecs UPLC C18 guard column (1.6 µm, 2.1 × 5 mm).The binary mobile phase was composed of acetonitrile as solvent A and deionized water with 10 mMAF as solvent B. Optimal run time for separation was 20 min using the following gradient programat a flow rate of 0.3 mL/min and a column oven temperature of 30 ◦C: isocratic 13% A at 0–0.5 min;0.5–2 min, linear from 13% to 16% A (v/v); 2–15 min, linear from 16% to 21% A (v/v); 15–16 min,linear from 21% to 60% A (v/v); 16–17 min, linear from 60% to 90% A (v/v); followed by columnre-equilibration using 13% A at 17–20 min. The injection volume was set to 2 µL.

2.6. MS Apparatus and Conditions

The MS detection was performed using an API 4000 Qtrap (AB Sciex, Foster City, CA, USA)equipped with an electrospray ionization (ESI) interface, and operated in negative ion mode withmultiple reaction monitoring (MRM). Turbo V ion source parameters were common to all analytes,as follows: the capillary voltage was −4.5 kV, and the source temperature was at 550 ◦C. The curtaingas and collision gas setting were 30 psi and 10 psi, respectively. The pressure for nebulization gas andvaporization gas setting were 55 psi. The declustering potential (DP) and collision energy (CE) wereoptimized for each analyte.

2.7. Data Processing

Evaluation of the different polysaccharides from genus Ephedra could be facilitated usingmultivariate statistical analysis. PCA was applied to visualize the data structure, classify the samplesaccording to their components and detect corresponding markers. The PCA was carried out usingSPSS version 16.0 (SPSS, Inc., Chicago, IL, USA).

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3. Results and Discussion

3.1. Derivatization and MS Conditions

In order to achieve a fast and accurate sample preparation, a simplified PMP-derivatizationprocedure was developed. The PMP reaction mechanism was illustrated in Figure 1A. This modifiedprocedure was characterized by extreme simplification without using any liquid–liquid extractionand concentrated steps by comparison with our previous reports [3]. The resulting solution wasdirectly filtered and diluted with deionized water before UPLC-ESI−-TQ-MS/MS. Furthermore,the comparative experiments between before and after liquid–liquid extraction showed that thesimplified workflow did not affect the chromatographic separation and MS signal intensities at all.This simplified sample preparation was applied successfully in the current study.

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3. Results and Discussion

3.1. Derivatization and MS Conditions

In order to achieve a fast and accurate sample preparation, a simplified PMP-derivatization procedure was developed. The PMP reaction mechanism was illustrated in Figure 1A. This modified procedure was characterized by extreme simplification without using any liquid–liquid extraction and concentrated steps by comparison with our previous reports [3]. The resulting solution was directly filtered and diluted with deionized water before UPLC-ESI−-TQ-MS/MS. Furthermore, the comparative experiments between before and after liquid–liquid extraction showed that the simplified workflow did not affect the chromatographic separation and MS signal intensities at all. This simplified sample preparation was applied successfully in the current study.

Figure 1. (A) Condensation reaction with PMP; (B) characteristic fragmentation pathways of bis-PMP derivatives of monosaccharides in negative ESI-MS/MS mode. Peaks were as follows: 1, Man; 2, GlcN; 3, ManN; 5, Rib; 6, Rha; 8, GalN; 11, GlcUA; 12, GalUA; 14, GlcNAc; 16, D2-Glc; 17, Glc; 18, GalNAc; 19, Gal; 20, Ara; 21, Fuc.

Regarding MS detection, carbohydrate PMP derivatives could be monitored under both ESI− and ESI+ modes. In the present work, much less noise was observed in the ESI− than ESI+ modes. Furthermore, the [M − H]− precursor ions were more intense for all analytes than [M + H]+ and [M + Na]+, so [M − H]− precursor ions were used for the detection of the carbohydrate PMP derivatives. Taking collision-induced dissociations of deprotonated PMP-labeled monosaccharides as examples, peaks 1–3, 5, 6, 8, 11, 12, 14, 16 and 17–21 produced similar fragmentation patterns. The typical ESI− fragmentation pathway is illustrated in Figure 1B. The [M − H]− precursor first experienced the loss of a PMP unit to afford a [M − PMP − H]− ion, which further produced α-cleavage to give characteristic [M′ − H]− ions at m/z 214, 215 or 256. This [M′ − H]− was considered to be a characteristic product ion based on the fact that this fragment was always stable with prominent intensity when using a specific collision energy. Therefore, the experimental MRM transition used those intact deprotonated m/z values of precursors (Q1) and characteristic fragments [M′ − H]− as product ions (Q3) for accurate detection of bis-PMP derivatives of monosaccharides–3, 5, 6, 8, 11, 12, 14 and 16–21. As a contrast,

Figure 1. (A) Condensation reaction with PMP; (B) characteristic fragmentation pathways of bis-PMPderivatives of monosaccharides in negative ESI-MS/MS mode. Peaks were as follows: 1, Man; 2, GlcN;3, ManN; 5, Rib; 6, Rha; 8, GalN; 11, GlcUA; 12, GalUA; 14, GlcNAc; 16, D2-Glc; 17, Glc; 18, GalNAc;19, Gal; 20, Ara; 21, Fuc.

Regarding MS detection, carbohydrate PMP derivatives could be monitored under both ESI−

and ESI+ modes. In the present work, much less noise was observed in the ESI− than ESI+ modes.Furthermore, the [M − H]− precursor ions were more intense for all analytes than [M + H]+ and[M + Na]+, so [M−H]− precursor ions were used for the detection of the carbohydrate PMP derivatives.Taking collision-induced dissociations of deprotonated PMP-labeled monosaccharides as examples,peaks 1–3, 5, 6, 8, 11, 12, 14, 16 and 17–21 produced similar fragmentation patterns. The typical ESI−

fragmentation pathway is illustrated in Figure 1B. The [M−H]− precursor first experienced the loss ofa PMP unit to afford a [M − PMP − H]− ion, which further produced α-cleavage to give characteristic[M′ − H]− ions at m/z 214, 215 or 256. This [M′ − H]− was considered to be a characteristic production based on the fact that this fragment was always stable with prominent intensity when usinga specific collision energy. Therefore, the experimental MRM transition used those intact deprotonatedm/z values of precursors (Q1) and characteristic fragments [M′ −H]− as product ions (Q3) for accuratedetection of bis-PMP derivatives of monosaccharides—3, 5, 6, 8, 11, 12, 14 and 16–21. As a contrast,

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bis-PMP derivatives of oligosaccharides 4, 7, 9, 10, 13 and 15 showed more complex fragmentationmechanism in ESI−-MS/MS mode, so the most intense product ion was chosen as Q3.

Finally, the optimal DP and CE values for all MRM transitions (1–22) are summarized in Table 1.Another advantage of the current method in comparison with traditional HPLC-UV methods is that itcould effectively eliminate the interference of the PMP ion peak using UPLC-ESI−-TQ-MS/MS withMRM detection.

Table 1. Mass spectrometry parameters used for analysis of targeted saccharides and internalstandards (IS).

Peaks Original Mw PMP Derivatives (m/z) Q1 (m/z) Q3 (m/z) DP (V) CE (V)

1 180.2 [M − H]− 509.1 215.1 −49.2 −28.12 215.6 [M − H]− 508.2 214.0 −65.9 −29.83 215.6 [M − H]− 508.4 213.7 −64.5 −26.04 1153.0 [M − H]− 1481.9 1308.0 −96.4 −35.15 150.1 [M − H]− 478.7 215.0 −58.4 −29.96 164.2 [M − H]− 493.2 214.9 −60.6 −29.07 990.9 [M − H]− 1319.7 869.8 −99.5 −61.28 215.6 [M − H]− 508.2 213.8 −67.9 −33.29 828.7 [M − H]− 1157.7 707.6 −90.3 −51.710 666.6 [M − H]− 995.6 214.8 −80.8 −52.611 212.2 [M − H]− 523.2 215.0 −55.9 −30.312 194.1 [M − H]− 523.1 215.0 −57.5 −47.413 504.4 [M − H]− 833.6 215.3 −98.4 −46.814 221.2 [M − H]− 550.2 256.2 −63.9 −32.915 360.3 [M − H]− 671.5 214.8 −61.2 −34.116 182.2 [M − H]− 511.2 214.6 −63.7 −28.017 180.2 [M − H]− 509.0 214.7 −59.9 −26.118 221.2 [M − H]− 550.3 256.0 −69.4 −34.119 180.2 [M − H]− 509.2 215.0 −65.0 −29.920 150.1 [M − H]− 479.0 215.1 −48.4 −33.920 150.1 [M − H]− 479.1 215.0 −63.1 −28.121 164.2 [M − H]− 493.1 215.1 −54.5 −30.422 126.1 [M − H]− 455.4 173.1 −60.9 −14.1

Peaks 1–3, 5, 6, 8, 11, 12, 15 and 16–21 were the same as in Figure 1. Other peaks are as follows: 4, maltoheptaose;7, maltohexaose; 9, maltopentaose; 10, maltotetraose; 13, maltotriose; 15, lactose; 22, 5-HMF.

3.2. Quantification and Validation

The chromatographic conditions were optimized to achieve the best resolution of varioussaccharides of interest within a short analysis. Some carbohydrate PMP derivatives might havedifferent structures, but have the same precursor ion and daughter ion. So it is difficult to fully separatethem by common HPLC techniques. Thus, UPLC-ESI−-TQ-MS/MS was explored to separate anddetect carbohydrates. To establish an ideal separation, the chromatographic parameters involvingcolumn types, additives of mobile phases, flow rates and column temperatures were optimized.

3.2.1. Effects of Columns

The UPLC method was developed to achieve a possible separation of various carbohydratePMP derivatives of interest. The result showed that some tested PMP derivatives were inadequatelyseparated (2 and 3, 5 and 6) by BEH C18 (2.1 × 100 mm, 1.7 µm) or available at a lower MS responseintensity (1, 2, 3, 8, 11, 12 and 16–19) using the HSS T3 column (2.1 mm × 100 mm, 1.8 µm) and ACEC18 (150 × 2.1mm, 3 µm) or a longer retention time up to 20–30 min (16–19) employing a HSS T3column (2.1 mm × 150 mm, 1.8 µm) and BEH C18 (2.1 × 100 mm, 1.7 µm). Therefore, a cortecs UPLCC18 (2.1 × 100 mm, 1.6 µm) column was used in this work to detect PMP derivatives 1–22. It wasfound that this specific column could provide high column efficiency and good MS response whileretaining a relatively short run time.

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3.2.2. Effects of Additives

To obtain chromatograms with good resolution and high intensity, various additives such asacetic acid, FA, and AF were investigated in the pre-experiment, and AF was shown to give thebest separation and peak shape. The effects of additive AF concentrations (8 mM, 10 mM, 15 mMand 10 mM AF with 0.1% FA) were also investigated on resolutions and MS response intensities.The results showed the moderate 10 mM AF showed the highest MS intensity for most of the peakstested. If 10 mM AF with a lower pH was applied, it seemed to make some peaks co-eluted, such as1–3 and 5–8. Therefore, 10 mM AF was used for further, optimized steps.

3.2.3. Effects of Column Temperature and Flow Rate

The effects of different column temperatures (25 ◦C, 30 ◦C and 35 ◦C) were assayed. Generally,all carbohydrate PMP derivatives tested were eluted to be a sharp peak at column temperatures of25–35 ◦C; there was no obvious effect on the peak symmetry and resolution of eluted analytes. However,peak responses were much better when the column temperature was applied at 30 ◦C. Therefore,the column temperature of 30 ◦C was used for further optimization steps. The effect of differentflow rates (0.2, 0.3 and 0.4 mL/min) has also been evaluated. After comprehensive consideration ofpeak resolutions, symmetries, retention time, and column pressures, 0.3 mL/min was selected as theoptimum flow rate.

Under the above optimum conditions, the MRM chromatograms of reference standards 1–22 aregiven in Figure 2, which shows the complete baseline separation of all corresponding isomericcarbohydrate PMP derivatives, including 1, 17 and 19 (Q1/Q3, 509/215), 2, 3, and 18 (Q1/Q3,508/214), 5 and 20 (Q1/Q3, 479/215), 6 and 21 (Q1/Q3, 493/215), and 11 and 12 (Q1/Q3, 523/215).These targeted analytes include 14 monosaccharides (1–3, 5, 6, 8, 11, 12, 14, 17–21), one internalstandard compound (16), six oligosaccharides (4, 7, 9, 10, 13 and 15) and one degraded product (22).Although some peaks such as 1, 2, 5, 6, 10–14 and 16–18 were not fully separated in total ionchromatography (Supplementary materials Figure S1), they were still accurately quantified dueto the presence of different MRM selections. It is worth mentioning that this is the first example onsimultaneous analysis of eight neutral sugars (1, 5, 6, 16, 17, 19–21), two uronic acids (11 and 12),three amino sugars (2, 3 and 8), two acetyl amino sugars (14 and 17), six oligosaccharides (4, 7, 9, 10,13 and 15), and one degradation product (22) using UPLC-ESI−-TQ-MS/MS.

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3.2.2. Effects of Additives

To obtain chromatograms with good resolution and high intensity, various additives such as acetic acid, FA, and AF were investigated in the pre-experiment, and AF was shown to give the best separation and peak shape. The effects of additive AF concentrations (8 mM, 10 mM, 15 mM and 10 mM AF with 0.1% FA) were also investigated on resolutions and MS response intensities. The results showed the moderate 10 mM AF showed the highest MS intensity for most of the peaks tested. If 10 mM AF with a lower pH was applied, it seemed to make some peaks co-eluted, such as 1–3 and 5–8. Therefore, 10 mM AF was used for further, optimized steps.

3.2.3. Effects of Column Temperature and Flow Rate

The effects of different column temperatures (25 °C, 30 °C and 35 °C) were assayed. Generally, all carbohydrate PMP derivatives tested were eluted to be a sharp peak at column temperatures of 25–35 °C; there was no obvious effect on the peak symmetry and resolution of eluted analytes. However, peak responses were much better when the column temperature was applied at 30 °C. Therefore, the column temperature of 30 °C was used for further optimization steps. The effect of different flow rates (0.2, 0.3 and 0.4 mL/min) has also been evaluated. After comprehensive consideration of peak resolutions, symmetries, retention time, and column pressures, 0.3 mL/min was selected as the optimum flow rate.

Under the above optimum conditions, the MRM chromatograms of reference standards 1–22 are given in Figure 2, which shows the complete baseline separation of all corresponding isomeric carbohydrate PMP derivatives, including 1, 17 and 19 (Q1/Q3, 509/215), 2, 3, and 18 (Q1/Q3, 508/214), 5 and 20 (Q1/Q3, 479/215), 6 and 21 (Q1/Q3, 493/215), and 11 and 12 (Q1/Q3, 523/215). These targeted analytes include 14 monosaccharides (1–3, 5, 6, 8, 11, 12, 14, 17–21), one internal standard compound (16), six oligosaccharides (4, 7, 9, 10, 13 and 15) and one degraded product (22). Although some peaks such as 1, 2, 5, 6, 10–14 and 16–18 were not fully separated in total ion chromatography (Supplementary materials Figure S1), they were still accurately quantified due to the presence of different MRM selections. It is worth mentioning that this is the first example on simultaneous analysis of eight neutral sugars (1, 5, 6, 16, 17, 19–21), two uronic acids (11 and 12), three amino sugars (2, 3 and 8), two acetyl amino sugars (14 and 17), six oligosaccharides (4, 7, 9, 10, 13 and 15), and one degradation product (22) using UPLC-ESI−-TQ-MS/MS.

Figure 2. MRM ion chromatograms and 22 reference standards. Peaks 1–3, 5, 6, 8, 11, 12, 15 and 16–21 are the same as in Figure 1. Other peaks are as follows: 4, maltoheptaose; 7, maltohexaose; 9, maltopentaose; 10, maltotetraose; 13, maltotriose; 15, lactose; 22, 5-HMF.

Figure 2. MRM ion chromatograms and 22 reference standards. Peaks 1–3, 5, 6, 8, 11, 12, 15 and16–21 are the same as in Figure 1. Other peaks are as follows: 4, maltoheptaose; 7, maltohexaose;9, maltopentaose; 10, maltotetraose; 13, maltotriose; 15, lactose; 22, 5-HMF.

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3.3. Method Validations

The analytical procedure has been validated in order to confirm its reliability. Table 2 summarizesthe calibration curves, LOD and LOQ values of the carbohydrate PMP derivatives analyzed byUPLC-ESI−- TQ-MS/MS with MRM mode. Since it is difficult to be precise about sample preparationsdue to the presence of derivatization procedures, the internal standard method is used. Here syntheticD2-Glc was chosen to be the internal standard in terms of different Q1/Q3 MRM selection. All thepeaks showed good linearity (R2 > 0.99) in quite a wide concentration range. The LOD and LOQ weredetermined at a signal-to-noise (S/N) of about 3 and 10, respectively. The results showed that the LODsof monosaccharides and oligosaccharides were in the range of 3–48 nM and 12–780 nM, respectively,indicating that the sensitivity of the method was satisfactory. The intra-day and inter-day RSD valuesof the analytes were all less than 4.86%, implying good precision and reproducibility. For stabilitytesting, the same sample solution was analyzed with 21 analytes every 4 h over 24 h with RSD lessthan 4.03%, which indicated that the sample was stable over two days under experimental conditions.

Table 2. Summarization of calibration results, LOD, and LOQ values.

No. Calibration Curves R2 Linear Range (µM) LOD (nM) LOQ (nM)

1 y = 0.2634x + 0.1916 0.9938 0.02–49.60 3.00 8.002 y = 0.1721x + 0.0360 0.9982 0.19–49.60 24.00 58.003 y = 0.2135x + 0.2631 0.9931 0.05–49.60 12.00 34.004 y = 0.0002x + 0.0002 0.9995 3.10–49.60 780.00 2240.005 y = 0.4846x + 0.2466 0.9960 0.02–49.60 3.00 92.006 y = 0.1496x + 0.1560 0.9952 0.19–49.60 48.00 140.007 y = 0.0012 x + 0.0013 0.9938 0.78–49.60 193.00 640.008 y = 0.1692x + 0.2124 0.9920 0.05–49.60 12.00 48.009 y = 0.0042x + 0.0009 0.9985 0.19–49.60 97.00 260.00

10 y = 0.0325x − 0.0110 0.9952 0.10–49.60 12.00 34.0011 y = 0.0661 x + 0.0919 0.9955 0.05–49.60 24.00 48.0012 y = 0.0223x + 0.0342 0.9935 0.39–49.60 32.00 96.0013 y = 0.0144x − 0.0040 0.9967 0.10–49.60 48.00 16.0014 y = 0.1207x + 0.0956 0.9938 0.05–49.60 12.00 34.0015 y = 0.0317x − 0.0026 0.9989 0.02–49.60 48.00 14.0017 y = 0.3381x + 0.2201 0.9973 0.02–49.60 3.00 6.0018 y = 0.3737x + 0.1974 0.9954 0.02–49.60 12.00 36.0019 y = 0.6961x + 0.4415 0.9954 0.02–49.60 3.00 10.0020 y = 0.6961x + 0.4415 0.9954 0.02–49.60 3.00 10.0021 y = 0.3787x + 0.4894 0.9925 0.19–49.60 24.00 72.0022 y = 0.4953x − 0.2220 0.9972 0.05–49.60 2.00 5.00

Peaks 1–22 were the same as Table 1. Peak 16 was the internal standard compound.

Furthermore, recovery experiments were performed in order to investigate the accuracy ofthe method. Known amounts of carbohydrate solutes were added to the sample with sameprocedures including extraction and hydrolysis, and the resulting spiked sample was subjectedto the entire analytical sequence. The recovery rate of the method was 94.37%–104.37%, with RSDless than 4.23%, suggesting that the method is accurate and practical for the composition analysis inplant polysaccharides.

The occurrence of matrix effects in UPLC-ESI−-TQ-MS/MS is well known and has an importantimpact on the determination of the constituents in complex plant samples. A matrix effect was obtainedby relative recoveries: relative recoveries = (sample contents after adding− original contents)/contentsof standard solutions for adding. The relative recoveries for all compounds ranged between 94.94%and 100.75%, thus showing the minimal matrix suppression or enhancement of this method.

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3.4. Application to Real Samples

After PMP derivatization of Ephedra polysaccharide-treated enzymatic digestions, it was directlyinjected and separated under the optimum conditions described above. The results of multipleenzymatic digestions are shown in Supplementary materials Table S1. Figure 3A–E shows typicalMRM ion chromatograms of E. sinica root polysaccharides using UPLC-ESI−-TQ-MS/MS beforeand after multiple enzymatic digestions, respectively. When the α-amylase was used for thetreatment of polysaccharides from genus Ephedrae, Ara (20), Glc (17), Gal (19), Man (1), lactose (15),and maltotriose (13) were readily liberated (Figure 3A). The β-(1→3)-D-glucanase digestion couldproduce both Ara (20) and Glc (17) (Figure 3B), while the pectinase digestion enabled Ara (20),Glc (17), GlcUA (11), GalUA (12), and lactose (15) to be liberated from the core skeletons ofEphedrae polysaccharides (Figure 3C). Meanwhile, a large amount of Glc (17), Ara (20), lactose (15),and maltotriose (13) were freed using the cellulose digestion of Ephedra polysaccharides (Figure 3D).If the endo-1,4-β-xylanase was applied, the predominant compositional saccharides were detectedto be Ara (20), Glc (17), lactose (15), maltotriose (13), and maltotetraose (10) (Figure 3E). Note alsothat a tetrasaccharide (Glc1(α)→4Glc(α)→4Glc(α)→4Glc(α)) residue should be linked in the side chain.The above evidence showed that Ephedra polysaccharides from different species and parts (stems orroots) may possess similar structural characteristics. Therefore, it is difficult to directly classify anddiscriminate these polysaccharides from genus Ephedra by eye.

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3.4. Application to Real Samples

After PMP derivatization of Ephedra polysaccharide-treated enzymatic digestions, it was directly injected and separated under the optimum conditions described above. The results of multiple enzymatic digestions are shown in Supplementary materials Table S1. Figure 3A–E shows typical MRM ion chromatograms of E. sinica root polysaccharides using UPLC-ESI−-TQ-MS/MS before and after multiple enzymatic digestions, respectively. When the α-amylase was used for the treatment of polysaccharides from genus Ephedrae, Ara (20), Glc (17), Gal (19), Man (1), lactose (15), and maltotriose (13) were readily liberated (Figure 3A). The β-(1→3)-D-glucanase digestion could produce both Ara (20) and Glc (17) (Figure 3B), while the pectinase digestion enabled Ara (20), Glc (17), GlcUA (11), GalUA (12), and lactose (15) to be liberated from the core skeletons of Ephedrae polysaccharides (Figure 3C). Meanwhile, a large amount of Glc (17), Ara (20), lactose (15), and maltotriose (13) were freed using the cellulose digestion of Ephedra polysaccharides (Figure 3D). If the endo-1,4-β-xylanase was applied, the predominant compositional saccharides were detected to be Ara (20), Glc (17), lactose (15), maltotriose (13), and maltotetraose (10) (Figure 3E). Note also that a tetrasaccharide (Glc1(α)→4Glc(α)→4Glc(α)→4Glc(α)) residue should be linked in the side chain. The above evidence showed that Ephedra polysaccharides from different species and parts (stems or roots) may possess similar structural characteristics. Therefore, it is difficult to directly classify and discriminate these polysaccharides from genus Ephedra by eye.

Figure 3. Cont.

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Figure 3. Typical MRM ion chromatograms of E. sinica root polysaccharides treated by multiple enzymatic digestions. (A), α-amylase; (B), β-(1→3)-D-glucanase, (C), pectinase; (D), cellulose; (E), endo-1,4-β-xylanase. (a,b) were due to polysaccharide samples with and without enzymatic digestions, respectively.

3.5. Discrimination of Ephedra Polysaccharides Based on PCA

As shown in Figure 4A,B,D, 20 Ephedra polysaccharide samples from different species and plant parts were well distinguished using α-amylase, β-(1→3)-D-glucanase, and cellulose enzymatic digestions, respectively. It was noticeable that samples 1–20 were clustered into four groups, a–d, which were unambiguously identified as polysaccharides from E. sinica stem, E. sinica root, E. intermedia stem, and E. equisetina stem, respectively. The principal components of enzymatic digestions (endo-1,4-β-xylanase and pectinase) had a minor effect on the model. The typical PCA scores from endo-1,4-β-xylanase and pectinase digestion are illustrated in Figure 4C,E, respectively.

The PCA loadings (not shown) were used to identify the differential components accountable for the separation among different groups. Ara, Gal, Glc, lactose, Man, and maltotriose were the principal components in classifying these samples for the α-amylase digestion of Ephedra polysaccharides. The Ara and Glc may play essential roles in the discrimination of Ephedra polysaccharides treated by the β-(1→3)-D-glucanase digestion. Meanwhile, the lactose, maltotriose, Ara, GalUA and Glc could be considered marker components to differentiate these samples using the cellulose digestion. These results further indicated that Ephedra polysaccharides, which were digested by α-amylase, β-(1→3)-D-glucanase, and cellulose, produced characteristic saccharide fingerprints. Therefore, UPLC-ESI−-TQ-MS/MS coupled with PCA could be used for differentiating Ephedra polysaccharides attributed to different species and plant parts through specific enzymatic digestions.

Figure 3. Typical MRM ion chromatograms of E. sinica root polysaccharides treated by multipleenzymatic digestions. (A), α-amylase; (B), β-(1→3)-D-glucanase, (C), pectinase; (D), cellulose;(E), endo-1,4-β-xylanase. (a,b) were due to polysaccharide samples with and without enzymaticdigestions, respectively.

3.5. Discrimination of Ephedra Polysaccharides Based on PCA

As shown in Figure 4A,B,D, 20 Ephedra polysaccharide samples from different species andplant parts were well distinguished using α-amylase, β-(1→3)-D-glucanase, and cellulose enzymaticdigestions, respectively. It was noticeable that samples 1–20 were clustered into four groups,a–d, which were unambiguously identified as polysaccharides from E. sinica stem, E. sinica root,E. intermedia stem, and E. equisetina stem, respectively. The principal components of enzymaticdigestions (endo-1,4-β-xylanase and pectinase) had a minor effect on the model. The typical PCAscores from endo-1,4-β-xylanase and pectinase digestion are illustrated in Figure 4C,E, respectively.

The PCA loadings (not shown) were used to identify the differential components accountable forthe separation among different groups. Ara, Gal, Glc, lactose, Man, and maltotriose were the principalcomponents in classifying these samples for the α-amylase digestion of Ephedra polysaccharides.The Ara and Glc may play essential roles in the discrimination of Ephedra polysaccharides treatedby the β-(1→3)-D-glucanase digestion. Meanwhile, the lactose, maltotriose, Ara, GalUA and Glccould be considered marker components to differentiate these samples using the cellulose digestion.These results further indicated that Ephedra polysaccharides, which were digested by α-amylase,β-(1→3)-D-glucanase, and cellulose, produced characteristic saccharide fingerprints. Therefore,UPLC-ESI−-TQ-MS/MS coupled with PCA could be used for differentiating Ephedra polysaccharidesattributed to different species and plant parts through specific enzymatic digestions.

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Figure 4. PCA score plots of 20 polysaccharide samples from multiple enzymatic digestions. (A) α-amylase; (B) β-(1→3)-D-glucanase, (C) pectinase; (D) cellulose; (E) endo-1,4-β-xylanase. (a) E. sinica (stem); (b) E. sinica (root); (c) E. intermedia (stem); (d) E. equisetina (stem).

4. Conclusions

In this work, a reliable, simple, and sensitive UPLC-ESI−-TQ-MS/MS method was developed for the simultaneous analysis of 21 PMP derivatives characterized by the presence of seven neutral sugars, two uronic acids, three amino sugars, two acetyl amino sugars, six oligosaccharides, and one degradation product based on a solid core cortecs C18 column within 20 min. The proposed method

Figure 4. PCA score plots of 20 polysaccharide samples from multiple enzymatic digestions.(A) α-amylase; (B) β-(1→3)-D-glucanase, (C) pectinase; (D) cellulose; (E) endo-1,4-β-xylanase.(a) E. sinica (stem); (b) E. sinica (root); (c) E. intermedia (stem); (d) E. equisetina (stem).

4. Conclusions

In this work, a reliable, simple, and sensitive UPLC-ESI−-TQ-MS/MS method was developedfor the simultaneous analysis of 21 PMP derivatives characterized by the presence of seven neutral

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sugars, two uronic acids, three amino sugars, two acetyl amino sugars, six oligosaccharides, and onedegradation product based on a solid core cortecs C18 column within 20 min. The proposedmethod minimizes sample handling, permits high-throughput analysis, and has been successfullyapplied to analyze 20 Ephedra polysaccharide samples from different species and plant parts.Multivariate statistical analysis results indicated that the specific enzymatic digestions (α-amylase,β-(1→3)-D-glucanase, and cellulose) could be used for distinguishing these polysaccharides fromgenus Ephedra. UPLC-ESI−-TQ-MS/MS, coupled with enzymatic digestions and multivariate statisticalanalysis, may be a powerful and useful approach for quality evaluation of plant polysaccharides fromTraditional Chinese Medicine.

Supplementary Materials: Supplementary Materials are available online.

Acknowledgments: Our work was partly financially supported by the National Natural Science Foundation ofChina (81303215) and the Foundation for the Author of National Excellent Doctoral Dissertation of China (201367).

Author Contributions: H.-X.K., Y.-G.X. and B.-Y.Y. designed this experiment. T.-L.W., L.-M.S. and J.L. participatedin data analysis and wrote this paper. All authors read and approved the final manuscript.

Conflicts of Interest: The authors declare no conflict of interest.

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Sample Availability: Compounds 1–22 are available from the authors.

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